Thionin-D4E1 chimeric protein protects plants against bacterial infections

ABSTRACT

The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The United States government has rights in this invention pursuant toContract No. DE-AC52-06NA25396 between the United States Department ofEnergy and Los Alamos National Security, LLC for the operation of LosAlamos National Laboratory.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to a chimeric protein made from a combination ofthionin and D4E1. This invention also relates to genetically alteredplants that can express this chimeric protein, and the use of thechimeric protein to protect plants against bacterial infections.

Description of Related Art

Plants have developed multiple defense mechanisms to combat invadingpathogenic microorganisms. For example, plants will synthesizeantimicrobial compound such as antimicrobial peptides (AMPs), thioninsand defensins. See, Broekaert, et al., Critical Review in Plant Sci.16:297-323 (2007). Many AMPs have been identified from variousorganisms. AMPs are short peptides with broad spectrum antimicrobialactivity against bacteria and fungi. AMPs can damage a pathogen's cellmembrane by inhibiting chitin synthase or β-D-glucan synthase. See,DeLucca and Walsh, Antimicrobial Agent and Chemotherapy 43:1-11 (1999).D4E1, one such antimicrobial peptide which is also a synthetic peptide,has a β-sheet conformation in solution and during interaction with cellmembranes which results in lytic activity. See, DeLucca, et al., Can. J.Microbiol. 44:514-520 (1998). In order to optimize the activity againsttarget pathogens, chimeric protein constructions, with or withoutmodification of amino acid sequences, have been designed including theanti-fungal and anti-bacterial lactoferricin B derivatives (Marcos, etal., Annu. Rev. Phytopathol. 46:273-301 (2008), antifungal cecropin Aand cecropin A-mellitin derived peptides (Monroc, et al., Peptides27:2567-2574 (2006); Monroc, et al., Peptides 27:2575-2584 (2006); andCavallarín, et al., Mol. Plant Microbe Interact. 11:218-227 (1998)), andbactericidal cyclic decapeptide BPC194 series (Monroc, et al., Peptides27:2567-2574 (2006); and Monroc, et al., Peptides 27:2575-2584 (2006)),and antifungal hexapeptide PAF26 (López-García, et al., Mol. PlantMicrobe Interact. 13:837-846 (2000); and López-García, et al., Appl.Environ. Microbiol. 68:2453-2460 (2002).

Many studies have reported that the expression of naturally occurringpeptides and their analogs confer resistance to pathogens in transgenicplants including Arabidopsis (Lee, et al., Plant Physiol. 148:1004-1020(2008)), tobacco (Huang, et al., Phytopathology 87:494-499 (1997);Jaynes, et al., Plant Sci. 89:43-53 (1993); and Cary, et al., Plant Sci.154:171-181 (2000)), Chinese cabbage (Jung, et al., Plant Biotechnol.Rep. 6:39-46 (2012)), rice (Coca, et al., Planta 223:392-406 (2006), andImamura, et al. Transgenic Res. 19:415-424 (2010)), cotton (Rajasekaran,et al., Plant Biotechnol. J. 3:545-554 (2006)), tomato (Alan, et al.,Plant Cell Rep. 22:388-396 (2006), and Alan, et al., Plant Cell Rep.22:388-396 (2006)), potato (Osusky, et al., Transgenic Res. 13:181-190(2004)), pear (Reynoird, et al., Plant Sci. 149:23-31 (1999)), banana(Chakrabarti, et al., Planta 216:587-596 (2003)), and hybrid poplar(Mentag, et al., Tree Physiol. 23:405-411 (2003)).

Compared to naturally produced peptides, synthetic peptides such as D4E1show rapid biocontrol or biostatic ability against various fungal andbacterial pathogens at low concentrations and can be designed to benon-toxic to mammalian and other animal cells (Jaynes, et al., PeptideRes. 2:157-160 (1989). In addition, synthetic peptides are generallydesigned to resist degradation by fungal and plant proteases and showtarget specificity and increased efficacy (Broekaert, et al. (2007), andMontesinos, E, FEMS Microbio. Lett. 270:1-11 (2007)). Haemolyticactivity of synthetic peptide D4E1 was found to be very low (Jaynes, etal., Plant Sci. 89:43-54 (1993)). It has been demonstrated that the puresynthetic peptide D4E1 is inhibitory to growth of about 20 bacterial andfungal phytopathogens (Rajasekaran, et al., J. Agric. Food Chem.49:2799-2803 (2001)). In addition, transgenic tobacco plants expressingD4E1 demonstrated a significant reduction in fungal growth in vitro andin planta (Cary, et al., Plant Sci. 154:171-181 (2000)). A 50% to 90%reduction in the viability of Fusarium verticillioides and Verticilliumdahliae was reported when spores were incubated in crude proteinextracts of D4E1-transformed cotton compared to extracts fromGUS-transformed cotton (Rajasekaran, et al., Plant Biotechnol. J.3:545-554 (2005)). D4E1 is effective in killing Chlamydia trachomatisin-vitro (Ballweber, et al., Antimicrobial Agents and Chemotherapy 46:1,34-41 (2002)). Purified D4E1 is highly effective at killingAgrobacterium tumefaciens, Sinorhizobium meliloti, and Xanthomonas citrissp. citri, but shows little hemolysis of porcine erythrocytes (Stover,et al., J. Amer. Soc. Hort. Sci. 138:142-148 (2013)).

Thionins from barley leaf are toxic to phytopathogenic bacteria andfungi in-vitro (Bohlmann, et al., EMBO J. 7:1559-1566 (1988); Molina, etal., Plant Sci. 92:169-177 (1993)). Thionins are small basic peptidescontaining between approximately 44 and approximately 47 amino acids andcontain a conserved cysteine-rich domain with toxic and antimicrobialproperties. Thionins are classified into two groups, a/b-thionins andc-thionins, based on their 3-D structure (Pelegrini and Franco, Int. J.Biochem. Cell. Bio. 37:2239-2253 (2005)). Thionins are postulated toinduce the opening of pores on the pathogen-cell membranes, allowingescape of potassium and calcium ions from pathogens' cell(s) (Pelegriniand Franco (2005); Oard, S. V., Biochim. Biophys. Acta 1808:1737-1745(2011)). It has been showed that a thionin gene from barley seedincreased resistance to Pseudomonas syringae when overproduced intransgenic tobacco plants (Carmona, et al., Plant J. 3:457-462 (1993)).Overexpression of the Arabidopsis thionin thi2.1 gene in Arabidopsisplants resulted in enhanced resistance to Fusarium oxysporum (Epple, etal., Plant Cell 9:509-520 (1997). Transgenic rice plants overproducingoat thionin displayed enhanced resistance to the bacterial diseasescaused by Burkholderia plantarii and B. glumae (Iwai, et al., Mol. PlantMicrobe Interact. 15:837-846 (2002)). Likewise, transgenic sweet potatooverproducing barley α-hordothionin had increased resistance to blackrot disease caused by Ceratocystis fimbriata (Muramoto, et al., PlantCell Rep. 31:987-97 (2012)).

To improve the efficacy of these anti-microbial agents, plantstransformed to express chimeric proteins containing two differentproteins or peptides have been assessed. In a recent study, grape plantswere transformed to express a chimeric protein containing humanneutrophil elastase and cecropin B. These transgenic grape plants hadresistance against Xylella fastidiosa ssp. fastidiosa which causesPierce disease in grapevines. See, Dandekar, et al., Proc. Nat. Acad.Sci. 109:10 3721-3725 (2012). These finding are important because X.fastidiosa is a Gram-negative bacterial pathogen with a wide range ofplant hosts of economic importance.

Huanglongbing (HLB) (also called “citrus greening”) and citrus cankercause serious diseases that threaten the Florida citrus industry. Thecausative agents of HLB are Candidatus Liberibacter africanus (CLaf),Candidatus Liberibacter asiaticus (CLas), and Candidatus Liberibacteramericanus (CLam). The bacteria are transmitted from plant to plant viaAsian citrus psyllid (Diaphorina citri), the African citrus psyllid(Trioza erytreae), and, perhaps, other hemipteran insects that feed fromcitrus plants' vascular tissue. Citrus canker is caused by Xanthomonascitri ssp. citri (Xcc). Xcc are spread by wind and perhaps insects andenter into citrus plants via wounds or other openings in the bark. Thesediseases are devastating the citrus industry in Florida because of noeffective treatment currently exists. Furthermore, both diseases and thecausative pathogens are spreading to other parts of the U.S. and othercitrus-producing countries.

A need exists for a method to prevent and/or treat these diseases incitrus plants. A need also exists to prevent and/or treat bacterialdiseases in plants. This invention involves genetically altered citrusplants that can produce a chimeric protein having broad-spectrumantimicrobial activity against gram-negative bacteria which can conferresistance to both citrus greening and canker diseases in citrus plants.This invention also involves other genetically altered plants expressingthis chimeric protein and that have resistance to diseases caused bybacteria.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of this invention to have a chimeric protein that has afirst domain, a second domain, and a third domain. It is another objectof this invention that the first domain is a thionin or pro-thionin, thesecond domain is D4E1 or pro-D4E1, and the third domain is a peptidelinker which separates the first domain from the second domain such thatthe first domain and the second domain can each fold into itsappropriate three-dimensional shape and retains its bacterial activity.It is another object of this invention that the peptide linker ranges inlength between approximately three amino acids and approximatelyforty-four amino acids. It is another object of this invention that thefirst domain and the second domain are positioned within this chimericprotein such that either the first domain or the second domain is at theamino terminus of the chimeric protein with the other domain (seconddomain or first domain, respectively) is located at the carboxylterminus of the chimeric protein. It is another object of this inventionthat this chimeric protein, when expressed in genetically alteredplants, kills bacteria and protects the genetically altered plant fromdiseases caused by bacteria.

It is another object of this invention that, when the first domain ofthis chimeric protein is located at the amino terminal of the chimericprotein, the first domain can be a thionin or pro-thionin from a plant(such as, but not limited to a citrus plant or Nicotiana benthamiana) orcan be an artificial thionin or pro-thionin which has been optimized(optimized thionin or optimized pro-thionin). It is another object ofthis invention that, when the first domain is located at the aminoterminal of the chimeric protein, the second domain is located at thecarboxyl terminal of the chimeric protein and is D4E1. It is anotherobject of this invention that the third domain is a peptide linker whichseparates the first domain from the second domain such that the firstdomain and the second domain can each fold into its appropriatethree-dimensional shape and retains its bacterial activity. It isanother object of this invention that the first domain has the aminoacid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 20, or SEQ ID NO: 22. It is a further object of thisinvention that the second domain has the amino acid sequence of SEQ IDNO: 2. It is yet another object of this invention that the peptidelinker has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 39, SEQID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42. It is another object of thisinvention that this chimeric protein, when expressed in geneticallyaltered plants, kills bacteria and protects the genetically alteredplant from diseases caused by bacteria.

It is an object of this invention that, when the first domain of thischimeric protein is located at the carboxyl terminus of the chimericprotein, the first domain is a thionin from a plant (such as, but notlimited to a citrus plant or Nicotiana benthamiana) or can be anartificial thionin which has been optimized (optimized thionin). It isanother object of this invention that, when the first domain is locatedat the carboxyl terminal of the chimeric protein, the second domain islocated at the amino terminus of the chimeric protein and is either D4E1or pro-D4E1. It is another object of this invention that the thirddomain is a peptide linker which separates the first domain from thesecond domain such that the first domain and the second domain can eachfold into its appropriate three-dimensional shape and retains itsbacterial activity. It is another object of this invention that thethionin (first domain) has the amino acid of SEQ ID NO: 14, SEQ ID NO:20, or SEQ ID NO: 22. It is a further object of this invention that thesecond domain, D4E1 or pro-D4E1, has the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 62. It is another object of this invention that thepeptide linker has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO:39, or SEQ ID NO: 54. It is another object of this invention that thischimeric protein, when expressed in genetically altered plants, killsbacteria and protects the genetically altered plant from diseases causedby bacteria.

It is an object of this invention to have a chimeric protein that has afirst domain at the amino terminus of the chimeric protein, a seconddomain at the carboxyl terminus of the chimeric protein, and a thirddomain that is a peptide linker that separates the first domain and thesecond domain such that the first domain and the second domain can eachfold into its appropriate three-dimensional shape and retains itsbacterial activity. It is another object of this invention that thefirst domain is a thionin or pro-thionin, and the second domain is D4E1.It is another object of this invention that the chimeric protein has theamino acid sequence of SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 24, SEQID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 67, SEQ ID NO: 68,SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO:73, or SEQ ID NO: 74. It is another object of this invention that thischimeric protein, when expressed in genetically altered plants, killsbacteria and protects the genetically altered plant from diseases causedby bacteria.

It is an object of this invention to have a chimeric protein that has afirst domain at the carboxyl terminus of the chimeric protein, a seconddomain at the amino terminus of the chimeric protein, and a third domainthat is a peptide linker that separates the first domain and the seconddomain such that the first domain and the second domain can each foldinto its appropriate three-dimensional shape and retains its bacterialactivity. It is another object of this invention that the first domainis a thionin, and the second domain is D4E1 or pro-D4E1. It is anotherobject of this invention that the chimeric protein has an amino acidsequence of SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:77, or SEQ ID NO: 78.

It is a further object of this invention to have one or morepolynucleotides that encodes the chimeric proteins of this invention. Itis yet another object of this invention to have an expression vectorthat contains a promoter operably linked to one or more polynucleotidesthat encodes the chimeric proteins. It is a further object of thisinvention to have a genetically altered plant or part thereof and itsprogeny that contains one or more of the polynucleotides of thisinvention. It is an object of this invention to have a flower, seed, orpollen of this genetically altered plant. It is yet another object ofthis invention to have a genetically altered plant cell or tissueculture of genetically altered plant cells that contain one or morepolynucleotides encoding the chimeric proteins of this inventionoperably linked to a promoter.

It is an object of this invention to have a method of constructing agenetically altered plant or part thereof having increased resistance tobacterial infections compared to a non-genetically altered plant or partthereof. It is a further object of this invention that the methodinvolves introducing one or more polynucleotides encoding one or more ofthe chimeric proteins of this invention into a plant or part thereof toprovide or generate a genetically altered plant or part thereof andselecting a genetically altered plant or part thereof that expresses thechimeric protein and that the expressed chimeric protein hasanti-bacterial activity. This chimeric protein has a first domain, asecond domain, and a third domain such that the first domain can bethionin or pro-thionin, the second domain can be D4E1 or pro-D4E1, andthe third domain is a peptide linker that separates the first domainfrom the second domain such that the first domain and the second domaincan each fold into its appropriate three-dimensional shape and retainsits bacterial activity, and such that the peptide linker ranges inlength between approximately three amino acids and approximatelyforty-four amino acids. It is a further object of the invention thatintroducing the one or more polynucleotides encoding the one or morechimeric proteins of this invention occurs via introgression ortransforming the plant with an expression vector containing the one ormore polynucleotides operably linked to a promoter. It is an optionalobject of this invention that, when the first domain is located at theamino terminus of the chimeric protein, it has the amino acid sequenceof SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:20, or SEQ ID NO: 22; and that, when the second domain is located at thecarboxyl terminus of the chimeric protein, it has the amino acidsequence of SEQ ID NO: 2; and that the peptide linker has the amino acidsequence of SEQ ID NO: 10, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,or SEQ ID NO: 42. It is another optional object of this invention that,when the second domain is located at the amino terminus of the chimericprotein, it has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:62; and that, when the first domain is located at the carboxyl terminusof the chimeric protein, it has an amino acid sequence of SEQ ID NO: 14,SEQ ID NO: 20, or SEQ ID NO: 22; and that the peptide linker has theamino acid sequence of SEQ ID NO: 10, SEQ ID NO: 39, or SEQ ID NO: 54.It is another object of this invention that the polynucleotide encodes achimeric protein that has the amino acid sequence of SEQ ID NO: 8, SEQID NO: 18, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ IDNO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or SEQ ID NO: 78. It is anoptional object of this invention that the polynucleotide has the DNAsequence of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61.

It is another object of this invention to have a method of enhancing aplant's resistance to bacterial diseases by transforming the plant orpart thereof with one or more polynucleotides that encode one or more ofchimeric proteins of this invention to generate a genetically alteredplant or part there of such that the genetically altered plant or partthereof produces the chimeric protein which kills bacteria that causebacterial diseases. This chimeric protein has a first domain, a seconddomain, and a third domain, such that the first domain can be thionin orpro-thionin, the second domain can be D4E1 or pro-D4E1, and the thirddomain is a peptide linker that separates the first domain from thesecond domain such that the first domain and the second domain can eachfold into its appropriate three-dimensional shape and retains itsbacterial activity, and such that the peptide linker ranges in lengthbetween approximately three amino acids and approximately forty-fouramino acids. It is an optional object of this invention that, when thefirst domain is located at the amino terminus of the chimeric protein,it has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 20, or SEQ ID NO: 22; and that, when thesecond domain is located at the carboxyl terminus of the chimericprotein, it has the amino acid sequence of SEQ ID NO: 2; and that thepeptide linker has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42. It is anotheroptional object of this invention that, when the second domain islocated at the amino terminus of the chimeric protein, it has the aminoacid sequence of SEQ ID NO: 2 or SEQ ID NO: 62; and that, when the firstdomain is located at the carboxyl terminus of the chimeric protein, ithas an amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 20, or SEQ IDNO: 22; and that the peptide linker has the amino acid sequence of SEQID NO: 10, SEQ ID NO: 39, or SEQ ID NO: 54. Optionally, thepolynucleotide encodes a chimeric protein that has the amino acidsequence of SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 67, SEQ IDNO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,or SEQ ID NO: 78. It is an optional object of this invention that thepolynucleotide has the DNA sequence of SEQ ID NO: 7, SEQ ID NO: 17, SEQID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:59, or SEQ ID NO: 61.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the amino acid sequences of Nicotiana benthamianapro-thionin (SEQ ID NO: 4), citrus pro-thionin (SEQ ID NO: 16), andoptimized pro-thionin (SEQ ID NO: 6).

FIG. 2 shows diagrams for the three binary vector pBINPLUS/ARScontaining the desired polynucleotides encoding the heterologousproteins (D4E1, “thionin” is optimized pro-thionin, and “chimera” isoptimized pro-thionin-linker 1-D4E1). In FIG. 2, “LB” is left border;“Ubi3-p” is the Ubi3 promoter; “NPTII” is neomycin phosphotransferasegene (which confers kanamycin resistance); “Ubi3-t” is Ubi3 terminator;“D35S-p” is double CaMV 35S promoter; “Nos-t” is nos terminator; and“RB” is right border. Thin arrows indicate position of PCR primers toconfirm gene integration located between D35S promoter and nosterminator regions. Thick arrow heads indicate locations of primers forRT-PCR and RT-qPCR located in target genes and nos terminator regions.

FIG. 3A and FIG. 3B show two gels loaded with the RT-PCR amplificationof total RNA from six genetically altered N. benthamiana plants (lanes2, 6, A, B, C, and D) expressing the heterologous chimeric protein(optimized pro-thionin-linker 1-D4E1), molecular weight marker (lane M),and genetically altered N. benthamiana plant expressing GUS (lane G).FIG. 3A shows the 300 bp RT-PCR fragment for the chimeric protein. FIG.3B shows the 500 bp RT-PCR fragment for N. benthamiana EF1α (NbEF1α).FIG. 3C and FIG. 3D show two gels loaded with the RT-PCR amplificationof total RNA from six genetically altered N. benthamiana plants (lanes3, 4, 5, 7, 9, and 11) expressing the heterologous optimized pro-thioninprotein, molecular weight marker (lane M), and genetically altered N.benthamiana plant expressing GUS (lane G). FIG. 3C shows the 220 bpRT-PCR fragment for the optimized pro-thionin protein. FIG. 3D shows the500 bp RT-PCR fragment for NbEF1α. FIG. 3E and FIG. 3F show two gelsloaded with the RT-PCR amplification of total RNA from six geneticallyaltered N. benthamiana plants (lanes 1, 2, 7, 8, 9, and 10) expressingthe heterologous D4E1 protein, molecular weight marker (lane M), andgenetically altered N. benthamiana plant expressing GUS (lane G). FIG.3E shows the 220 bp RT-PCR fragment for the D4E1 protein. FIG. 3F showsthe 500 bp RT-PCR fragment for NbEF1α.

FIG. 4A, FIG. 4B, FIG. 4C show quantitation analysis of RNA levels indifferent genetically altered N. benthamiana lines: chimeric proteinoptimized pro-thionin-linker 1-D4E1 (FIG. 4A), optimized pro-thinion(FIG. 4B), and D4E1 (FIG. 4C). RT-qPCR is performed to compare geneexpression level of the independent genetically altered N. benthamianalines. Relative gene expression of target gene is normalized to theexpression of the NbEF1α.

FIG. 5A illustrates the relative gene expression levels for optimizedpro-thionin in eight genetically altered Carrizo lines using RT-qPCR.FIG. 5B illustrates the gene expression levels for chimeric protein(optimized pro-thionin-linker 1-D4E1) in five genetically alteredCarrizo lines using RT-qPCR. FIG. 5C illustrates the gene expressionlevels for D4E1 protein in four genetically altered Carrizo lines usingRT-qPCR.

FIG. 6 shows genetically altered orange plants expressing eitheroptimized pro-thionin-linker 1-D4E1 or only D4E1 and negative controlplants (nontransformed) after exposure to X. citri.

DETAILED DESCRIPTION OF THE INVENTION

In order to protect citrus plants from two devastating diseases, thisinvention involves the generation of a chimeric protein and geneticallyaltered citrus plants to express the chimeric protein. This chimericprotein has the ability to protect citrus plants from Xanthomonas citri(the causative agent of canker) and from the Candidatus Liberibacterspecies (the causative agent of HBL or citrus greening disease).Genetically altered plants expressing/producing this chimeric proteinexhibit no symptoms or much reduced symptoms of the diseases caused bythese bacteria when exposed to the bacteria. It is also within the scopeof this invention that any plant can be genetically altered to expressthe chimeric proteins described herein, and that the genetically alteredplant will be protected from gram-negative bacterial infections becausethe expressed chimeric proteins kill the gram-negative bacteria thatinfect the plant. It is also within the scope of this invention that thechimeric proteins kill gram-positive bacteria.

Thus, this invention also covers the method of preventing or treatinggram-negative or gram-positive bacterial diseases in a plant withaltered DNA (and the plant's progeny) by transforming the plant (orotherwise altering the DNA of plant) with one or more polynucleotideswhich encode one or more of the chimeric proteins described herein andallow the plant to produce the one or more chimeric proteins. Thechimeric protein will kill gram-negative bacteria after thegram-negative bacteria infect the plant. The invention also includes amethod of enhancing a plant's resistance to bacterial diseases bytransforming a plant (or otherwise altering the DNA of plant) with oneor more polynucleotides encoding one or more chimeric proteins describedherein such that the plant containing the heterologous DNA produces thechimeric protein, and the chimeric protein kills bacteria that causebacterial diseases after the bacteria infect the plant.

In one embodiment of this invention, the chimeric protein has threedomains, a first domain containing thionin or pro-thinion, a seconddomain containing D4E1, and a third domain containing a peptide linker.This peptide linker domain is a set of amino acids that separate thefirst domain from the second domain and prevents steric inhibitionbetween the first domain and the second domain. In one embodiment, thefirst domain (thionin or pro-thinion) is at the amino terminus of thechimeric protein, and the second domain (D4E1) is at the carboxylterminus. In another embodiment, the second domain (D4E1 or pro-D4E1) isat the amino terminus of the chimeric protein while the first domain(thionin) is at the carboxyl terminus.

When thionin is located at the amino terminus of the chimeric protein,it is encoded as a pro-protein (pro-thionin) which contains an aminoacid signal sequence (the exact number of amino acids in the signalsequence can vary by organism). The signal sequence assists in thetrafficking of pro-thionin to the endoplasmic reticulum or a cellularvesicle. Not wishing to be bound to a particular hypothesis, it isbelieved that the signal sequence is cleaved off pro-thionin prior to,during, or after passage of pro-thionin through the lipid membrane toyield mature thionin. See, Romero, et al., Eur. J. Biochem. 243:202-8(1997). When thionin (first domain) is located at the carboxyl terminusof the chimeric protein, thionin (first domain) does not contain anamino acid signal sequence. However, the chimeric protein still cancontain an amino acid signal sequence at the amino terminus of thechimeric protein as described below. The thionin (or pro-thionin) can bea thionin (or pro-thionin) that exists in a plant (and more specificallyin N. benthamiana or a citrus plant), or an optimized thionin (oroptimized pro-thionin) as described below. The third domain, the peptidelinker, can be Linker 1 (SEQ ID NO: 10), Linker 2 (SEQ ID NO: 39),Linker 3 (SEQ ID NO: 40), Linker 4 (SEQ ID NO: 41), or Linker 5 (SEQ IDNO: 42).

When the second domain is located at the amino terminus of the chimericprotein and first domain is located at the carboxyl terminus, a signalsequence upstream of D4E1 can be used to traffic the chimeric protein tothe endoplasmic reticulum or cellular vesicle. This signal sequence canbe the signal sequence for the optimized pro-thionin or citrus'pro-thionin or any other signal sequence from a plant. Optimizedpro-thionin's signal sequence is 31 amino acids long (SEQ ID NO: 12) andhas the DNA sequence of SEQ ID NO: 11. The DNA sequence and amino acidsequence for citrus' pro-thionin signal sequence is in SEQ ID NO: 37 andSEQ ID NO: 38, respectively. Thus, when the second domain is located atthe amino terminus of the chimeric protein, it can be D4E1 or pro-D4E1.The chimeric protein containing D4E1 or pro-D4E1 at the amino terminalcan contain, in the third domain, Linker 1 (SEQ ID NO: 10), Linker 2(SEQ ID NO: 39), or Linker 6 (SEQ ID NO: 54). Further, the first domain,located at the carboxyl terminus of the chimeric protein can be thionin(from N. benthamiana (SEQ ID NO: 19) or a citrus plant (SEQ ID NO: 22))or optimized thionin (SEQ ID NO: 14). To be clear, the DNA sequenceencoding the chimeric protein containing D4E1 or pro-D4E1 at the aminoterminus does not encode pro-thionin (or optimized pro-thionin).Instead, the DNA sequence encodes mature thionin (or mature optimizedthionin) (i.e., without the amino acid signal sequence). For thepurposes of this invention, when a signal sequence is adjacent to D4E1,it is referred to as “pro-D4E1” because the signal sequence amino acidsare removed during post-translational processing to generate the matureD4E1.

In one embodiment, the first domain of the chimeric protein contains“optimized thionin” (or optimized pro-thionin). The nucleotide and aminoacid sequence of the optimized thionin differs from thionin naturallypresent in plants. One difference between optimized thionin and thioninfrom some plants is the addition of five amino acids at the carboxylterminus of the protein. Optimized thionin also includes some amino acidsubstitutions, the number and location depending on the plant thioninsequence to which it is being compared. One substitution involvesreplacing an arginine to a phenylalanine. Optimized thionin has (i) anincrease in anti-bacterial activity and (ii) a reduced toxicity to theplant compared to wild-type thionin (or “non-optimized” thionin). SeeFIG. 1 for a comparison of the amino acid sequences of N. benthamianapro-thionin, citrus pro-thionin (obtained from Citrus sinensis), andoptimized pro-thionin. The first 31 amino acids for N. benthamianapro-thionin and optimized pro-thionin and the first 29 amino acids forcitrus pro-thionin are the signal sequence amino acids for therespective proteins. In another embodiment, the first domain of thechimeric protein is citrus thionin (or citrus pro-thionin). In yetanother embodiment, the first domain of the chimeric protein is N.benthamiana thionin (or N. benthamiana pro-thionin).

The third domain (peptide linker) can range from two amino acids long toapproximately 50 amino acids long in one embodiment. In anotherembodiment, the linker domain ranges from three amino acids toapproximately 44 amino acids. In another embodiment, the linker domainranges from four amino acids to approximately 44 amino acids. In yetanother embodiment, the linker domain is four, five, six, seven, eight,nine, ten, eleven, twelve, or thirteen amino acids long. A linker shouldbe sufficiently flexible such that the first domain and the seconddomain of the chimeric protein are able to fold into their correctthree-dimensional shape and retain their activity. The linker should notreduce solubility of the chimeric protein, and can, in certaincircumstances, enhance solubility of the chimeric protein. The linkerand the chimeric protein containing the particular linker should benon-toxic to the genetically altered plant expressing the chimericprotein. In one embodiment, the linker can assist in generating asynergistic effect between the first domain and second domain of thechimeric protein. Non-limiting examples of linkers are provided in Table1, infra. As demonstrated in the examples, below, genetically alteredplants expressing the chimeric protein have better/greater antibacterialactivity than genetically altered plants expressing either optimizedthionin (or thionin) only or D4E1 only. This greater antibacterialactivity is surprising and unexpected. It is believed that thisbetter/greater antibacterial activity results from the synergisticeffect of having D4E1 and optimized thionin (or thionin) attached via apeptide linker (and thus exist as “one” protein).

This invention covers the various chimeric proteins having the differentconfigurations disclosed herein, their amino acid sequences, and theirDNA sequences. This invention also covers genetically altered plantswhich carry heterologous DNA encoding one or more of the chimericproteins described herein and which can express the one or more chimericproteins encoded by the heterologous DNA. As such, expression vectorswhich contain heterologous DNA (encoding a chimeric protein) operablylinked to a promoter are also covered in this invention. To help in thegenerations of the expression vectors and genetically altered plantscontaining the heterologous DNA encoding a chimeric protein, the DNAsequences encoding the chimeric proteins disclosed herein may includeadditional nucleotides at the 5′ and 3′ end of the polynucleotide whichassist in generating the expression vectors and/or assist in thetranscription of the polynucleotide into mRNA and/or translation of themRNA into the chimeric protein.

In the examples below, plants are genetically altered to express eitherD4E1, optimized thionin (or optimized pro-thionin), or one of thechimeric proteins discussed herein. This invention also covers plantswhich contain these heterologous DNA via introgression. To assist inunderstanding the examples and inventions described herein, Table 1lists the various proteins or polypeptides and their DNA and/or aminoacid sequence listing number. One can use the information in Table 1 togenerate a chimeric protein containing linker 2, linker 3, linker 4,linker 5, or linker 6 with the desired thionin instead of optimizedthionin and with D4E1.

TABLE 1 Amino acid Protein or polypeptide DNA sequence # sequence # D4E1SEQ ID NO: 1 SEQ ID NO: 2 Pro-D4E1 SEQ ID NO: 61 SEQ ID NO: 62 N.benthamiana pro-thionin SEQ ID NO: 3 SEQ ID NO: 4 N. benthamiana thioninSEQ ID NO: 19 SEQ ID NO: 20 Citrus pro-thionin (Citrus sinensis) SEQ IDNO: 15 SEQ ID NO: 16 Citrus thionin (Citrus sinensis) SEQ ID NO: 21 SEQID NO: 22 Optimized pro-thionin SEQ ID NO: 5 SEQ ID NO: 6 Optimizedthionin SEQ ID NO: 13 SEQ ID NO: 14 Chimeric protein (N. benthamiana SEQID NO: 23 SEQ ID NO: 24 pro-thionin - linker 1 - D4E1) Chimeric protein(N. benthamiana SEQ ID NO: 25 SEQ ID NO: 26 thionin - linker 1 - D4E1)Chimeric protein (optimized pro- SEQ ID NO: 7 SEQ ID NO: 8 thionin -linker 1 - D4E1) Chimeric protein (optimized SEQ ID NO: 17 SEQ ID NO: 18thionin - linker 1 - D4E1) Chimeric protein (citrus pro- SEQ ID NO: 27SEQ ID NO: 28 thionin - linker 1 - D4E1) Chimeric protein (citrus SEQ IDNO: 29 SEQ ID NO: 30 thionin - linker 1 - D4E1) Chimeric protein(optimized pro- SEQ ID NO: 67 thionin - linker 2 - D4E1) Chimericprotein (optimized pro- SEQ ID NO: 68 thionin - linker 3 - D4E1)Chimeric protein (optimized pro- SEQ ID NO: 69 thionin - linker 4 -D4E1) Chimeric protein (optimized pro- SEQ ID NO: 70 thionin - linker5 - D4E1) Chimeric protein (optimized SEQ ID NO: 71 thionin - linker 2 -D4E1) Chimeric protein (optimized SEQ ID NO: 72 thionin - linker 3 -D4E1) Chimeric protein (optimized SEQ ID NO: 73 thionin - linker 4 -D4E1) Chimeric protein (optimized SEQ ID NO: 74 thionin - linker 5 -D4E1) Chimeric protein (D4E1 - linker 1 - SEQ ID NO: 31 SEQ ID NO: 32 N.benthamiana thionin) Chimeric protein (D4E1 - linker 1 - SEQ ID NO: 33SEQ ID NO: 34 optimized thionin) Chimeric protein (D4E1 - linker 1 - SEQID NO: 35 SEQ ID NO: 36 citrus thionin) Chimeric protein (pro-D4E1 - SEQID NO: 55 SEQ ID NO: 56 linker 1 - N. benthamiana thionin) Chimericprotein (pro-D4E1 - SEQ ID NO: 57 SEQ ID NO: 58 linker 1 - optimizedthionin) Chimeric protein (pro-D4E1 - SEQ ID NO: 59 SEQ ID NO: 60 linker1 - citrus thionin) Chimeric protein (D4E1 - linker 2 - SEQ ID NO: 75optimized thionin) Chimeric protein (D4E1 - linker 6 - SEQ ID NO: 76optimized thionin) Chimeric protein (pro-D4E1 - SEQ ID NO: 77 linker 2 -optimized thionin) Chimeric protein (pro-D4E1 - SEQ ID NO: 78 linker 6 -optimized thionin) Signal sequence for optimized SEQ ID NO: 11 SEQ IDNO: 12 pro-thionin and N. benthamiana pro-thionin Signal sequence forcitrus SEQ ID NO: 37 SEQ ID NO: 38 pro-thionin Linker 1 SEQ ID NO: 9 SEQID NO: 10 Linker 2 SEQ ID NO: 39 Linker 3 SEQ ID NO: 40 Linker 4 SEQ IDNO: 41 Linker 5 SEQ ID NO: 42 Linker 6 SEQ ID NO: 54

The chimeric peptides of this invention are effective against a widerange of gram-negative plant-pathogenic bacteria. Examples of diseasescaused by gram-negative bacteria include, but not be limited, to crowngall (caused by Agrobacterium spp.); Pierce's Disease, Almond Scorch,Coffee Scorch, and Citrus Variegated Chlorosis (caused by Xylella spp.);citrus canker and various bacterial blights, spots and wilts (caused byXanthomonas spp.); blasts, leaf spots, cankers and diebacks (caused byPseudomonas spp.); and blights and soft-rots (caused by Erwinia spp.).In addition to CLas, CLam, and CLaf that cause HLB disease in citrusplants, other Candidatus Liberibacter species infect various plants andare pathogenic. For example, Candidatus Liberibacter psyllaurous infectspotato and tomato plants, and Candidatus Liberibacter solanacearuminfect potato plants causing zebra chip disease. Other bacteria thatinfect plants and cause disease are known in the art. Geneticallyaltered plants expressing the chimeric protein(s) describe herein haveenhanced protection against these diseases and the pathogens causing thediseases.

Because this invention involves production of genetically altered plantsand involves recombinant DNA techniques, the following definitions areprovided to assist in describing this invention. The terms “isolated”,“purified”, or “biologically pure” as used herein, refer to materialthat is substantially or essentially free from components that normallyaccompany the material in its native state or when the material isproduced. In an exemplary embodiment, purity and homogeneity aredetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A nucleicacid or particular bacteria that are the predominant species present ina preparation is substantially purified. In an exemplary embodiment, theterm “purified” denotes that a nucleic acid or protein that gives riseto essentially one band in an electrophoretic gel. Typically, isolatednucleic acids or proteins have a level of purity expressed as a range.The lower end of the range of purity for the component is about 60%,about 70% or about 80% and the upper end of the range of purity is about70%, about 80%, about 90% or more than about 90%.

The term “nucleic acid” as used herein, refers to a polymer ofribonucleotides or deoxyribonucleotides. Typically, “nucleic acid”polymers occur in either single- or double-stranded form, but are alsoknown to form structures comprising three or more strands. The term“nucleic acid” includes naturally occurring nucleic acid polymers aswell as nucleic acids comprising known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Exemplary analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, andpeptide-nucleic acids (PNAs). “DNA”, “RNA”, “polynucleotides”,“polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleicacid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acidfragment”, and “isolated nucleic acid fragment” are used interchangeablyherein.

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). Estimates are typically derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions), the complementary (or complement)sequence, and the reverse complement sequence, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); andRossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because the aminoacid sequences of D4E1, thionin, pro-thionin, optimized thionin,optimized pro-thionin, linker 1, linker 2, linker 3, linker 4, linker 5,linker 6, and the chimeric proteins are described, one can chemicallysynthesize a polynucleotide which encodes these polypeptides/chimericproteins. Because of the degeneracy of nucleic acid codons, one can usevarious different polynucleotides to encode identical polypeptides.Table 2, infra, contains information about which nucleic acid codonsencode which amino acids.

TABLE 2 Amino acid Nucleic acid codons Ala/A GCT, GCC, GCA, GCG Arg/RCGT, CGC, CGA, CGG, AGA, AGG Asn/N AAT, AAC Asp/D GAT, GAC Cys/CTGT, TGC Gln/Q CAA, CAG Glu/E GAA, GAG Gly/G GGT, GGC, GGA, GGG His/HCAT, CAC Ile/I ATT, ATC, ATA Leu/L TTA, TTG, CTT, CTC, CTA, CTG Lys/KAAA, AAG Met/M ATG Phe/F TTT, TTC Pro/P CCT, CCC, CCA, CCG Ser/STCT, TCC, TCA, TCG, AGT, AGC Thr/T ACT, ACC, ACA, ACG Trp/W TGG Tyr/YTAT, TAC Val/V GTT, GTC, GTA, GTG

In addition to the degenerate nature of the nucleotide codons whichencode amino acids, alterations in a polynucleotide that result in theproduction of a chemically equivalent amino acid at a given site, but donot affect the functional properties of the encoded polypeptide, arewell known in the art. “Conservative amino acid substitutions” are thosesubstitutions that are predicted to interfere least with the propertiesof the reference polypeptide. In other words, conservative amino acidsubstitutions substantially conserve the structure and the function ofthe reference protein. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine or histidine,can also be expected to produce a functionally equivalent protein orpolypeptide. Table 3 provides a list of exemplary conservative aminoacid substitutions. Conservative amino acid substitutions generallymaintain (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a beta sheet or alpha helicalconformation, (b) the charge or hydrophobicity of the molecule at thesite of the substitution, and/or (c) the bulk of the side chain.

TABLE 3 Amino Acid Conservative Substitute Ala Gly, Ser Arg His, Lys AsnAsp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His GluAsp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, ValLys Arg, Gln, Glu Met Ile, Leu Phe His, Leu, Met, Trp, Tyr Ser Cys, ThrThr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Oligonucleotides and polynucleotides that are not commercially availablecan be chemically synthesized e.g., according to the solid phasephosphoramidite triester method first described by Beaucage andCaruthers, Tetrahedron Letts. 22:1859-1862 (1981), or using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.12:6159-6168 (1984). Other methods for synthesizing oligonucleotides andpolynucleotides are known in the art. Purification of oligonucleotidesis by either native acrylamide gel electrophoresis or by anion-exchangeHPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, organism,nucleic acid, protein or vector, has been modified by the introductionof a heterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells may express genes thatare not found within the native (non-recombinant or wild-type) form ofthe cell or express native genes that are otherwise abnormallyexpressed—over-expressed, under-expressed or not expressed at all.

The terms “transgenic”, “transformed”, “transformation”, and“transfection” are similar in meaning to “recombinant”.“Transformation”, “transgenic”, and “transfection” refer to the transferof a polynucleotide into the genome of a host organism or into a cell.Such a transfer of polynucleotides can result in genetically stableinheritance of the polynucleotides or in the polynucleotides remainingextra-chromosomally (not integrated into the chromosome of the cell).Genetically stable inheritance may potentially require the transgenicorganism or cell to be subjected for a period of time to one or moreconditions which require the transcription of some or all of thetransferred polynucleotide in order for the transgenic organism or cellto live and/or grow. Polynucleotides that are transformed into a cellbut are not integrated into the host's chromosome remain as anexpression vector within the cell. One may need to grow the cell undercertain growth or environmental conditions in order for the expressionvector to remain in the cell or the cell's progeny. Further, forexpression to occur the organism or cell may need to be kept undercertain conditions. Host organisms or cells containing the recombinantpolynucleotide can be referred to as “transgenic” or “transformed”organisms or cells or simply as “transformants”, as well as recombinantorganisms or cells.

A genetically altered organism is any organism with any change to itsgenetic material, whether in the nucleus or cytoplasm (organelle). Assuch, a genetically altered organism can be a recombinant or transformedorganism. A genetically altered organism can also be an organism thatwas subjected to one or more mutagens or the progeny of an organism thatwas subjected to one or more mutagens and has changes in its DNA causedby the one or more mutagens, as compared to the wild-type organism (i.e,organism not subjected to the mutagens). Also, an organism that has beenbred to incorporate a mutation into its genetic material is agenetically altered organism. For the purposes of this invention, theorganism is a plant.

The term “vector” refers to some means by which DNA, RNA, a protein, orpolypeptide can be introduced into a host. The polynucleotides, protein,and polypeptide which are to be introduced into a host can betherapeutic or prophylactic in nature; can encode or be an antigen; canbe regulatory in nature; etc. There are various types of vectorsincluding virus, plasmid, bacteriophages, cosmids, and bacteria.

An expression vector is nucleic acid capable of replicating in aselected host cell or organism. An expression vector can replicate as anautonomous structure, or alternatively can integrate, in whole or inpart, into the host cell chromosomes or the nucleic acids of anorganelle, or it is used as a shuttle for delivering foreign DNA tocells, and thus replicate along with the host cell genome. Thus, anexpression vector are polynucleotides capable of replicating in aselected host cell, organelle, or organism, e.g., a plasmid, virus,artificial chromosome, nucleic acid fragment, and for which certaingenes on the expression vector (including genes of interest) aretranscribed and translated into a polypeptide or protein within thecell, organelle or organism; or any suitable construct known in the art,which comprises an “expression cassette”. In contrast, as described inthe examples herein, a “cassette” is a polynucleotide containing asection of an expression vector of this invention. The use of thecassettes assists in the assembly of the expression vectors. Anexpression vector is a replicon, such as plasmid, phage, virus, chimericvirus, or cosmid, and which contains the desired polynucleotide sequenceoperably linked to the expression control sequence(s).

A polynucleotide sequence is operably linked to an expression controlsequence(s) (e.g., a promoter and, optionally, an enhancer) when theexpression control sequence controls and regulates the transcriptionand/or translation of that polynucleotide sequence.

Transformation and generation of genetically altered monocotyledonousand dicotyledonous plant cells is well known in the art. See, e.g.,Weising, et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Pat. No.5,679,558; Agrobacterium Protocols, ed: Gartland, Humana Press Inc.(1995); and Wang, et al. Acta Hort. 461:401-408 (1998). The choice ofmethod varies with the type of plant to be transformed, the particularapplication and/or the desired result. The appropriate transformationtechnique is readily chosen by the skilled practitioner.

Exemplary transformation/transfection methods available to those skilledin the art include, but are not limited to: direct uptake of foreign DNAconstructs (see, e.g., EP 295959); techniques of electroporation (see,e.g., Fromm et al., Nature 319:791 (1986)); and high-velocity ballisticbombardment with metal particles coated with the nucleic acid constructs(see, e.g., Kline, et al., Nature 327:70 (1987) and U.S. Pat. No.4,945,050). Specific methods to transform heterologous genes intocommercially important crops (to make genetically altered plants) arepublished for rapeseed (De Block, et al., Plant Physiol. 91:694-701(1989)); sunflower (Everett, et al., Bio/Technology 5:1201 (1987));soybean (McCabe, et al., Bio/Technology 6:923 (1988), Hinchee, et al.,Bio/Technology 6:915 (1988), Chee, et al., Plant Physiol. 91:1212-1218(1989), and Christou, et al., Proc. Natl. Acad. Sci USA 86:7500-7504(1989)); rice (Hiei, et al., Plant J. 6:271-282 (1994)), and corn(Gordon-Kamm, et al., Plant Cell 2:603-618 (1990), and Fromm, et al.,Biotechnology 8:833-839 (1990)). Other known methods are disclosed inU.S. Pat. Nos. 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,262,316;and 5,569,831.

One exemplary method includes employing Agrobacterium tumefaciens orAgrobacterium rhizogenes as the transforming agent to transferheterologous DNA into the plant. Agrobacterium tumefaciens-meditatedtransformation techniques are well described in the scientificliterature. See, e.g., Horsch, et al. Science 233:496-498 (1984), andFraley, et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983). Typically, aplant cell, an explant, a meristem or a seed is infected withAgrobacterium tumefaciens transformed with the expressionvector/construct which contains the heterologous nucleic acid operablylinked to a promoter. Under appropriate conditions known in the art, thetransformed plant cells are grown to form shoots, roots, and developfurther into genetically altered plants. In some embodiments, theheterologous nucleic acid can be introduced into plant cells, by meansof the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid istransmitted to plant cells upon infection by Agrobacterium tumefaciens,and is stably integrated into the plant genome. See, e.g., Horsch, etal. (1984), and Fraley, et al. (1983).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the desired transformed phenotype. Such regenerationtechniques rely on manipulation of certain phytohormones in a tissueculture growth medium, typically relying on a biocide and/or herbicidemarker which has been introduced together with the desired nucleotidesequences. Plant regeneration from cultured protoplasts is described inEvans et al., Protoplasts Isolation and Culture, in Handbook of PlantCell Culture, pp. 124-176, MacMillan Publishing Company, New York, 1983;and Binding, Regeneration of Plants, in Plant Protoplasts, pp. 21-73,CRC Press, Boca Raton, 1985. Regeneration can also be obtained fromplant callus, explants, organs, or parts thereof. Such regenerationtechniques are described generally in Klee, et al., Ann. Rev. of PlantPhys. 38:467-486 (1987).

This invention utilizes routine techniques in the field of molecularbiology. Basic texts disclosing the general methods of use in thisinvention include Green and Sambrook, 4th ed. 2012, Cold Spring HarborLaboratory; Kriegler, Gene Transfer and Expression: A Laboratory Manual(1993); and Ausubel et al., eds., Current Protocols in MolecularBiology, 1994—current, John Wiley & Sons. Unless otherwise noted,technical terms are used according to conventional usage. Definitions ofcommon terms in molecular biology maybe found in e.g., Benjamin Lewin,Genes IX, published by Oxford University Press, 2007 (ISBN 0763740632);Krebs, et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The term “plant” includes whole plants, plant organs, progeny of wholeplants or plant organs, embryos, somatic embryos, embryo-likestructures, protocorms, protocorm-like bodies (PLBs), and suspensions ofplant cells. Plant organs comprise, e.g., shoot vegetativeorgans/structures (e.g., leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g., bracts, sepals, petals, stamens,carpels, anthers and ovules), seed (including embryo, endosperm, andseed coat) and fruit (the mature ovary), plant tissue (e.g., vasculartissue, ground tissue, and the like) and cells (e.g., guard cells, eggcells, trichomes and the like). The class of plants that can be used inthe method of the invention is generally as broad as the class of higherand lower plants amenable to the molecular biology and plant breedingtechniques described herein, specifically angiosperms (monocotyledonous(monocots) and dicotyledonous (dicots) plants). It includes plants of avariety of ploidy levels, including aneuploid, polyploid, diploid,haploid and hemizygous. The genetically altered plants described hereincan be monocot crops, such as, sorghum, maize, wheat, rice, barley,oats, rye, millet, and triticale. The genetically altered plantsdescribed herein can also be dicot crops, such as apple, pear, peach,plum, orange, lemon, lime, grapefruit, pomegranate, olive, peanut,tobacco, etc. Also, the genetically altered plants (or plants withaltered genomic DNA) can be horticultural plants such as rose, marigold,primrose, dogwood, pansy, geranium, etc. In some embodiments, thegenetically altered plants are citrus plants. In other embodiments, thegenetically altered plants are N. benthamiana or tobacco plants.

Once a genetically altered plant has been generated, one can breed itwith a wild-type plant and screen for heterozygous F1 generation plantscontaining the genetic change present in the parent genetically alteredplant. Then F2 generation plants can be generated which are homozygousfor the genetic alteration. These heterozygous F1 generation plants andhomozygous F2 plants, progeny of the original genetically altered plant,are considered genetically altered plants, having the altered genomicmaterial from the genetically altered parent plant.

After one obtains a genetically altered plant expressing the chimericprotein, one can efficiently breed the genetically altered plant withother plants containing desired traits. One can use molecular markers(i.e., polynucleotide probes) based on the sequence of the chimericprotein as described above to determine which offspring of crossesbetween the genetically altered plant and the other plant have thepolynucleotide encoding the chimeric protein. This process is known asMarker Assisted Rapid Trait Introgression (MARTI). Briefly, MARTIinvolves (1) crossing the genetically altered plant with a plant linehaving desired phenotype/genotype (“elite parent”) for introgression toobtain F1 offspring. The F1 generation is heterozygous for chimericprotein trait. (2) Next, an F1 plant is be backcrossed to the eliteparent, producing BC1F1 which genetically produces 50% wild-type and 50%heterozygote chimeric protein. (3) PCR using the polynucleotide probe isperformed to select the heterozygote genetically altered plantscontaining polynucleotide encoding the chimeric protein. (4) Selectedheterozygotes are then backcrossed to the elite parent to performfurther introgression. (5) This process of MARTI is performed foranother four cycles. (6) Next, the heterozygote genetically alteredplant is self-pollinated by bagging to produce BC6F2 generation. TheBC6F2 generation produces a phenotypic segregation ratio of 3 wild-typeparent plants to 1 chimeric protein genetically altered plant. (7) Oneselects genetically altered chimeric protein plants at the BC6F2generation at the seedling stage using PCR with the polynucleotide probeand can optionally be combined with phenotypic selection at maturity.These cycles of crossing and selection can be achieved in a span of 2 to2.5 years (depending on the plant), as compared to many more years forconventional backcrossing introgression method now in use. Thus, theapplication of MARTI using PCR with a polynucleotide probe significantlyreduces the time to introgress the chimeric protein genetic alterationinto elite lines for producing commercial hybrids. The final product isan inbred plant line almost identical (99%) to the original elitein-bred parent plant that is the homozygous for the polynucleotideencoding the chimeric protein.

The terms “approximately” and “about” refer to a quantity, level, valueor amount that varies by as much as 30%, or in another embodiment by asmuch as 20%, and in a third embodiment by as much as 10% to a referencequantity, level, value or amount. As used herein, the singular form “a”,“an”, and “the” include plural references unless the context clearlydictates otherwise. For example, the term “a bacterium” includes both asingle bacterium and a plurality of bacteria.

Having described the invention in general terms, below are examplesillustrating the generation and efficacy of the invention.

Example 1 Generation of Genetically Altered N. benthamiana

Genetically altered N. benthamiana plants are generated to assess theantibacterial efficacy of D4E1 (SEQ ID NO: 2), optimized thionin (SEQ IDNO: 14) or the chimeric protein, optimized thionin-linker 1-D4E1 (SEQ IDNO: 18). To improve the antibacterial activity of N. benthamiana thioninand to reduce its toxicity to N. benthamiana plants, in-silico assaysare performed which indicate that thionin's activity could be enhancedby changing the DNA sequence so that optimized thionin contains fiveadditional acidic amino acids compared to the wild-type N. benthamianathionin amino acid sequence. In addition, an arginine is substituted forphenylalanine to reduce plant toxicity in optimized thionin. See FIG. 1for a comparison of N. benthamiana pro-thionin amino acid sequence (SEQID NO: 4) and optimized pro-thionin amino acid sequence (SEQ ID NO: 6).The chimeric protein, optimized thionin-linker 1-D4E1 (SEQ ID NO: 18),contains linker 1 which is seven amino acids (peptide linker, thirddomain) (SEQ ID NO: 10) between the thionin domain (first domain) andthe D4E1 domain (second domain) to reduce steric hindrance of the twoactive domains of the chimeric protein and so that each domain retainsits antibacterial activity, thus generating a synergistic antibacterialactivity. As discussed above, optimized thionin and the chimericprotein, optimized thionin-linker 1-D4E1, are encoded and translated aspro-thionin and contain 31 amino acids (SEQ ID NO: 12) as a signalsequence which assists in trafficking the pro-protein to and through theendoplasmic reticulum or a vesicle. The signal sequence amino acids arecleaved off the pro-peptide to generate the mature protein. The DNA andamino acid sequences for optimized pro-thionin are in SEQ ID NO: 5 andSEQ ID NO: 6, respectively; and the DNA and amino acid sequences for thechimeric protein, optimized pro-thionin-linker 1-D4E1 are in SEQ ID NO:7 and SEQ ID NO: 8, respectively.

DNA2.0 (Menlo Park, Calif.) is hired to synthesize the DNA sequences forthe three proteins (D4E1 only, optimized pro-thionin, and optimizedpro-thionin-linker 1-D4E1 (chimeric protein or chimeria)) which are usedto generate the genetically altered N. benthamiana plants. To assist inthe generation of the appropriate plasmids and expression vectors, eachDNA sequence contains additional nucleotides at the 5′ end and 3′ end togenerate recognition sites for restriction endonucleases Sma1 and Kpn1,respectively. Thus the DNA sequence of Sma1-D4E1-Kpn1 is in SEQ ID NO:43. The DNA sequence of Sma1-optimized pro-thionin-Kpn1 is in SEQ ID NO:44. The DNA sequence of Sma1-optimized pro-thionin-linker 1-D4E1-Kpn1 isin SEQ ID NO: 45. DNA 2.0 supplies the chemically synthesizedpolynucletoides in individual pJ224 plasmids.

Each pJ224 plasmid is digested with Sma1 and Kpn1 per the manufacturer'ssuggested protocol (New England Biolabs, Ipswich, Mass.). The DNA fromeach reaction is run on an agrose gel, and the appropriate sized bands(for D4E1 approximately 320 bp, for pro-thionin approximately 520 bp,and chimera approximately 600 bp) are excised and then purified using agel purification kit (Qiagen, Valencia, Calif.). The purified DNA(desired polynucleotide) is then ligated to binary vector pBINPLUS/ARSwith T4 ligase (Promega Corp., Madison, Wis.) such that the desiredpolynucleotide is operably linked upstream (5′) to a double CaMV 35Spromoter (D35S) and downstream (3′) to Nos terminator sequence (Nos-t).As seen in FIG. 2, the pBINPLUS/ARS vector also contains, upstream ofthe D35S promoter, the Ubi3 promoter (Ubi3-p) operably linked toneomycin phosphotransferase gene (NPTII) which confers kanamycinresistance, which is then operably linked to Ubi3 terminator (Ubi3-t).In FIG. 2, thin arrows indicate the location of PCR primers to confirmDNA integration located between D35S promoter and nos terminatorregions. Thick arrow heads indicate the location of primers for RT-PCRand RT-qPCR in target genes and nos terminator regions, respectively.

The three separate ligation reactions are used, individually, totransform E. coli TOPO10 competent cells (Invitrogen, Carlsbad, Calif.).Transformed E. coli are streaked on plates, and positive bacterialclones are determined by colony PCR, followed by plasmid isolation, andsequencing to confirm the presence of plasmids containing the desiredsequences. The binary vectors carrying DNA encoding the desired peptidesare introduced into A. tumefaciens EHA105 by electroporation. The binaryvector pBINPLUS/ARS carrying GUS (β-D-glucuronidase) is also introducedinto A. tumefaciens EHA105 as negative control. Strains and vectors usedin this study are listed in Table 4.

TABLE 4 Plasmids Relevant characteristics Source pJ224 Cloning vector,Amp^(r) DNA 2.0 pBINPLUS/ARS-26 Binary vector carrying GUS, Kan^(r)USHRL pBINPLUS/ARS-D4E1 Binary vector carrying D4E1, Kan^(r) This studypBINPLUS/ARS- Binary vector carrying optimized This study thioninthionin*, Kan^(r) pBINPLUS/ARS- Binary vector carrying chimeric Thisstudy chimera protein**, Kan^(r) *contains DNA encoding optimizedpro-thionin **contains DNA encoding optimized pro-thionin - linker 1 -D4E1

Transformation of N. benthamiana is performed using theAgrobacterium-mediated leaf disk transformation method (Krugel, et al.,Chemoecology 12:177-183 (2002)). Leaf disks are cultured on Murashigeand Skoog (MS) medium with addition of 6-benzylaminopurine at 1 mg/L,0.1 mg/L naphthaleneacetic acid (NAA), 150 mg/L kanamycin (kan), and 200mg/L cefotaxime. The kan-resistant plants are selected and rooted in MSmedium containing NAA at 0.1 mg/L, 100 mg/L kan and 100 mg/L cefotaxime.The rooted plants are potted in a commercial soil mix for further testin a controlled green house. Most of the genetically altered N.benthamiana plants containing optimized pro-thionin(pBIN/ARSPLUS-thionin) are morphologically similar to non-transformed orgus transformed negative control plants except genetically altered N.benthamiana line thionin-4 which has long and narrow leaves. Geneticallyaltered N. benthamiana plants expressing the chimeric protein (optimizedpro-thionin-linker 1-D4E1) are morphologically similar tonon-transformed plants.

Over ten kanamycin-resistant transformed N. benthamiana plants for eachplasmid used for transformation (pBINPLUS/ARS-26, pBINPLUS/ARS-D4E1,pBINPLUS/ARS-thionin, and pBINPLUS/ARS-chimera) are obtained. Six plantsfor each group are selected for further analysis. To test if the T-DNAregion is integrated into the N. benthamiana genome, primers aredesigned to span from the D35S region to Nos terminator region. DNA fromeach genetically altered plant is individually isolated with plantDNeasy Plant kits (Qiagen, Valencia, Calif.) according to manufacturer'ssuggested protocol. The D35S promoter primer is5′-GACGCACAATCCCACTATCC-3′ (SEQ ID NO: 46); the Nos terminator primer is5′-TTTGCGCGCTATATTTTGTTT-3′ (SEQ ID NO: 47). The isolated plant genomicDNA is amplified using Taq polymerase (Epicenter, Madison, Wis.) andthese primers. The PCR reaction conditions are denature 95° C. for 3minutes and then 32 cycles of 95° C. for 30 seconds to denature, 53° C.for 30 seconds to anneal, and 72° C. for 1 minute for extension. The PCRproducts are run on 1% agrose gel and stained with ethidium bromide. Theexpected 350 bp, 500 bp, and 600 bp fragments are observed,respectively, for transgenic lines carrying pBINPLUS/ARS-D4E1,pBINPLUS/ARS-thionin, or pBINPLUS/ARS-chimera. Meanwhile, a 2,000 bpband is present in gus transformed control plant carryingpBINPLUS/ARS-26.

RT-PCR is performed on the genetically altered N. benthamiana plants toassess the expression levels of the heterologous DNA. Trizol reagent isused for RNA extraction according to the manufacture's instruction(Sigma-Aldrich, St. Louis, Mo.). Total RNA is quantified using Nanodrop(Thermo Fisher Scientific, Wilmington, Del.) and is treated with RQ1RNase-free DNase from Promega Corp (Madison, Wis.). DNase-treated RNA(˜1.5 μg) is used to synthesize first-strand cDNA with 0.5 μg of oligo(dT) primer and 1 μL of SuperScript® III reverse transcriptase in a 20μL reaction (Invitrogen, Carlsbad, Calif.). A negative control withoutthe reverse transcriptase is performed to verify the absence of genomicDNA contamination. PCR is performed as described above with cDNA astemplate and 26 cycles for all genetically altered lines [optimizedpro-thionin-linker 1-D4E1 (pBINPLUS/ARS-chimera); optimized pro-thionin(pBINPLUS/ARS-thionin), and D4E1 (pBINPLUS/ARS-D4E1)]. However, after 26cycles of RT-PCR with genetically altered N. benthamiana plant linescarrying the optimized pro-thionin-linker 1-D4E1 (pBINPLUS/ARS-chimera),no band or only a very faint band is amplified. So, RT-PCR is performedwith 32 cycles in the optimized pro-thionin-linker 1-D4E1 geneticallyaltered N. benthamiana plants (pBINPLUS/ARS-chimera).

FIG. 3A and FIG. 3B show two gels loaded with the 32 cycles RT-PCRamplification of total RNA from six genetically altered N. benthamianaplants (lanes 2, 6, A, B, C, and D) expressing the heterologous chimericprotein (optimized pro-thionin-linker 1-D4E1), molecular weight marker(lane M), and genetically altered N. benthamiana plant expressing GUS(lane G). FIG. 3A shows the 300 bp RT-PCR fragment for the chimericprotein. FIG. 3B shows the 500 bp RT-PCR fragment for N. benthamianaEF1α (NbEF1α). As evident in FIG. 3A, five genetically altered N.benthamiana lines containing the chimeric protein (optimizedpro-thionin-linker 1-D4E1; pBINPLUS/ARS-chimera) (chimera 2, 6, A, B andC) show stronger gene expression compare to genetically altered N.benthamiana chimera D line. Similar fragments are amplified with NbEF1αforward and reverse primers (SEQ ID NOs: 52 and 53) which serve as acontrol for cDNA input using the same RT-PCR protocol.

FIG. 3C and FIG. 3D show two gels loaded with the 26 cycles RT-PCRamplification of total RNA from six genetically altered N. benthamianaplants (lanes 3, 4, 5, 7, 9, and 11) expressing the heterologousoptimized pro-thionin protein, molecular weight marker (lane M), andgenetically altered N. benthamiana plant expressing GUS (lane G). FIG.3C shows the 220 bp RT-PCR fragment for optimized pro-thionin. FIG. 3Dshows the 500 bp RT-PCR fragment for NbEF1α. As evident in FIG. 3C, fivegenetically altered N. benthamiana optimized thionin lines (thionin 3,4, 5, 7 and 11) (pBINPLUS/ARS-thionin) show stronger gene expressioncompared to genetically altered N. benthamiana optimized thionin 9 line.Similar products are amplified with NbEF1α forward and reverse primers(SEQ ID NOs: 52 and 53) from these plants using the same RT-PCRprotocol.

FIG. 3E and FIG. 3F show two gels loaded with the 26 cycles RT-PCRamplification of total RNA from six genetically altered N. benthamianaplants (lanes 1, 2, 7, 8, 9, and 10) expressing the heterologous D4E1protein, molecular weight marker (lane M), and genetically altered plantexpressing GUS (lane G). FIG. 3E shows the 220 bp RT-PCR fragment forD4E1. FIG. 3F shows the 500 bp RT-PCR fragment for NbEF1α. Again asevident in FIG. 3E, D4E1 gene expression varies in different geneticallyaltered N. benthamiana lines. Strong PCR amplification is observed ingenetically altered N. benthamiana D4E1 line 8 and genetically alteredN. benthamiana D4E1 line 9; in contrast only a faint signal is seen forgenetically altered N. benthamiana D4E1 line 1 and genetically alteredN. benthamiana D4E1 line 2.

To further compare the gene expression level in these geneticallyaltered N. benthamiana lines, RT-qPCR is used to determined RNAabundance against internal control, NbEF1α. RT-qPCR is performed withBright Green PCR Master Mix (Sigma-Aldrich, St. Louis, Mo.) intriplicate using an ABI 7500 thermal cycler (Applied Biosystems, FosterCity, Calif.). Each 25 μL amplification reaction contains 12.5 μL ofBright Green PCR Master Mix, 250 nM of each forward primer and reverseprimer, and 2.0 μL of 1:10 diluted cDNA template. The following protocolis used: 95° C. for 2 minutes, 40 cycles of 30 seconds for denaturationat 95° C. and 30 seconds for extension at 60° C. For D4E1 N. benthamianatransformants (pBINPLUS/ARS-D4E1), forward primer is5′-CAAGGTGAGGTTGAGAGCTAAG-3′ (SEQ ID NO: 48) and reverse primer is5′-TCCTAGTTTGCGCGCTATATTT-3′ (SEQ ID NO: 49). For optimized pro-thioninN. benthamiana transformants (pBINPLUS/ARS-thionin) and chimeric proteinoptimized pro-thionin-linker 1-D4E1 transformants (pBINPLUS/ARS-chimera)forward primer is 5′-TTTCGCCGTAGATGCAGATG-3′ (SEQ ID NO: 50), andreverse primer is 5′-TCCTAGTTTGCGCGCTATATTT-3′ (SEQ ID NO: 51). NbEF1αis amplified and used to normalize the values as an internal controlusing forward primer 5′-GACCACTGAAGTTGGATCTGTTG-3′ (SEQ ID NO: 52) andreverse primer 5′-TAGCACCAGTTGGGTCCTTCTT-3′ (SEQ ID NO: 53). To comparethe transcript levels, a ratio of relative gene expression is calculatedfrom the 2^(ΔC) values of a sample versus a reference sample which haslowest 2^(ΔC) in the tested samples. ΔCT=CT (target gene)−CT (internalcontrol). The qPCR reactions are performed in triplicate and repeatedtwice times with similar results. RNA abundance varies between differentgenetically altered lines and within each group of genetically alteredlines.

Genetically altered N. benthamiana line chimera 6 (optimizedpro-thionin-linker 1-D4E1; pBIN/ARSPLUS-chimera) has the highest levelof gene expression compare to other genetically altered N. benthamianachimera lines (FIG. 4A). Genetically altered N. benthamiana linethionin-4 has over 25 fold expression compared to genetically altered N.benthamiana line thionin-9. Genetically altered N. benthamiana linesthionin-3 and thionin-11 also shows relatively high gene expression(FIG. 4B). For D4E1, genetically altered N. benthamiana lines D4E1-8 andD4E1-9 show higher expression levels compared to other geneticallyaltered N. benthamiana D4E1 lines (FIG. 4C).

Example 2 Pathogenicity Assays with Genetically Altered N. benthamiana

P. syringae ssp. tabaci can cause wildfire and angular leaf spot on N.benthamiana plants. P. syringae ssp. tabaci strains that produce thetoxin calledas tabtoxinin-β-lactam cause wildfire disease; in contrast,strains that do not produce the toxin produce only angular leaf spotsymptoms (Stover, et al. (2013)). Typically wildfire disease ischaracterized by small brown or black lesions surrounded by chlorotichalos caused by the toxin's mechanism of action. The angular leaf spotis characterized as larger brown or black lesions without chlorotichalos and has angular margins. The P. syringae ssp. tabaci producingonly angular leaf spots symptoms is used in this example.

P. syringae ssp. tabaci are obtained from Florida Department ofAgriculture and Consumer Services. Overnight cultures of P. syringaessp. tabaci are centrifuged and diluted to the following concentrationin sterile distilled water. Dilutions of 10², 10³, 10⁴, 10⁵ and 10⁶CFU/ml are determined by the standard plate-dilution method (Huang, etal. (1997)). Two leaves of each plant are inoculated with each of thefive bacterial inoculum level. The bacterial suspensions are infiltratedfrom the abaxial side into leaves of the genetically altered N.benthamiana plants (D4E1, optimized pro-thionin, optimizedpro-thionin-linker 1-D4E1 (chimeric protein), and GUS (negativecontrol)) using a syringe. Inoculated plants are incubated for 14 days.Disease development is scored and photographed. The experiment isrepeated three times with similar results.

Yellowing or necrotic lesions are first observed 6 day post infiltration(dpi). Symptoms continue to develop through the 14 days evaluationperiod. On 14 dpi, the gus transformed N. benthamiana lines (negativecontrol) show brown necrosis lesion at the concentration of 10⁶ CFU/mland yellowing and many brown lesion spots at 10³-10⁵ CFU/ml (see Table5). Genetically altered N. benthamiana lines expressing optimizedthionin (optimized pro-thionin) show remarkable resistance with onlyslight necrosis at the highest infiltration level of 10⁶ CFU/ml. A fewlesion spots are observed on all genetically altered N. benthamianalines expressing optimized thionin (see Table 5). Remarkably,genetically altered N. benthamiana line 3 expressing optimized thioninonly shows slight necrosis on the edge of the infiltrated zone withinfiltration at 10⁶ CFU/ml while a few small necrosis lesions areobserved with infiltration at 10³-10⁵ CFU/ml. Genetically altered N.benthamiana lines expressing the chimera (optimized pro-thionin-linker1-D4E1) show variation for disease development. Genetically altered N.benthamiana lines 2, 6 and B expressing chimera show less necrosisdevelopment. However genetically altered N. benthamiana lines A and Fexpressing the chimera only show moderate resistance compare to thenegative controls. At low infiltration levels (10⁴, 10³ and 10² CFU/ml),a few necrosis spots are observed (see Table 5). Genetically altered N.benthamiana lines expressing D4E1 (D4E1) only show slightly lessnecrosis compared to gus transformed N. benthamiana lines (negativecontrol) at low infiltration concentration of 10³ and 10² CFU/ml (seeTable 5). These results demonstrate that genetically altered N.benthamiana plants expressing optimized thionin or a chimeric protein ofoptimized thionin-linker 1-D4E1 significantly reduce infection caused byP. syringae ssp. tabaci, however genetically altered N. benthamianaplants expressing D4E1 had only slightly increased resistance to P.syringae ssp. tabaci.

TABLE 5 Genetically altered Infiltration Concentration (CFU/ml) N.benthamiana line 10² 10³ 10⁴ 10⁵ 10⁶ GUS-1 (negative) 15 26 N⁻ N⁻ N GUS-2 (negative) 5 22 N⁻ N⁻ N  Chimera-2 0 0 0 3 N⁻ Chimera-6 0 0 2 4 N⁻Chimera-A 2 10 15 27 N  Chimera-B 0 0 4 13 N⁻ Chimera-C 0 3 8 32 N⁻Chimera-D 0 19 33 N⁻ N  Thionin-3 0 1 1 2 N⁻ Thionin-4 0 2 4 11 N⁻Thionin-5 0 3 17 28 N⁻ Thionin-7 0 2 11 45 N⁻ Thionin-9 0 3 6 18 N⁻Thionin-11 0 2 3 9 N⁻ D4E1-1 0 8 38 N⁻ N  D4E1-2 2 12 44 N⁻ N  D4E1-7 06 32 N⁻ N  D4E1-8 0 6 26 N⁻ N  D4E1-9 0 8 19 N⁻ N  D4E1-10 0 7 35 N⁻ N “0” indicates no necrotic lesion observed. A digit other than “0”indicates the average number of small spot lesions per infiltration site(means of four infiltration sites). N⁻ indicates necrosis development. Nindicates brown necrosis lesions.

Example 3 Generation of Genetically Altered Orange Trees

Three sets of genetically altered orange plants are generated using thepolynucleotides described above and chemically synthesized by DNA 2.0(Menlo Park, Calif.). One polynucleotide (SEQ ID NO: 1) encodes for D4E1(SEQ ID NO: 2). The second polynucleotide (SEQ ID NO: 5) encodes foroptimized pro-thionin (SEQ ID NO: 6) which, during translation of mRNAinto the protein or during post-translation modification, the signalsequence amino acids (SEQ ID NO: 12) are removed to generate optimizedthionin (SEQ ID NO: 14). The third polynucleotide (SEQ ID NO: 7) encodesthe chimeric protein, optimized pro-thionin-linker 1-D4E1 (SEQ ID NO: 8)which, during translation of mRNA into the peptide or duringpost-translation modification, the signal sequence amino acids (SEQ IDNO: 12) are removed to generate the chimeric protein, optimizedthionin-linker 1-D4E1 (SEQ ID NO: 18). As discussed in Example 1 above,each DNA sequence contains additional nucleotides at the 5′ end and 3′end to generate recognition sites for restriction endonucleases Sma1 andKpn1, respectively (see SEQ ID NO: 43 (D4E1), SEQ ID NO: 44 (optimizedpro-thionin), and SEQ ID NO: 45 (optimized pro-thionin-linker 1-D4E1)).The pJ244 plasmids containing these polynucleotides are digested withSma1 and Kpn1 using the recommended protocol provided by supplier. (NewEngland Biolabs, Ipswich, Mass.) For each construct, the digested DNA isrun on an agrose gel, and the desired DNA band (approximately 320 bp forD4E1; approximately 520 bp for optimized thionin; and approximately 600bp for chimeric protein) is excised and purified. The plasmid pUSHRL-15is also digested with Sma1 and Kpn1 using manufacturer's recommendedprotocol. Then purified polynucleotide encoding each desired polypeptideare ligated with the Sma1, Kpn1 digested pUSHRL-15 in form pUSHRL-15D4E1(encoding D4E1), pUSHRL-15thionin (encoding pro-thionin), andpUSHRL-15chimeric protein (encoding optimized pro-thionin-linker1-D4E1), respectively. Similar to the pBINPLUS/ARS expression vectordescribed in Example 1 above, pUSHRL expression vector contains D35Spromoter upstream of (5′) and operably linked to the ligated desiredpolynucleotide, and Nos-t downstream of (3′) and operably linked to theligated desired polynucleotides. The pUSHRL expression vector alsocontains Ubi3-p operably linked to NPTII which confers kanamycinresistance, which is then operably linked to Ubi3-t, all upstream of theD35S.

Each of pUSHRL-15D4E1, pUSHRL-15thionin, and pUSHRL-15chimeric proteinare individually introduced into Agrobacterium tumefaciens strain EHA105 by electroporation, and the resulting recombinant bacteria are usedfor the transformation of citrus (Carrizo variety (C. sinensis×P.trifoliata)). The transformation protocol essentially follows Orbovicand Grosser (Citrus Methods in Molecular Biology 344:177-189 (2006))using epicotyl explants tissues that are suspended in a solution of A.tumefaciens and plated on selective media containing kanamycin.Kanamycin-resistant plants are selected and rooted in MS mediumcontaining NAA at 0.1 mg/L, 100 mg/L kan and 100 mg/L cefotaxime.Regenerated plantlets are rooted in-vitro and then established in thegreenhouse ex-vitro in commercial soil mix. Plants from eachtransformation are selected for RT-PCR and Southern blot assays toconfirm transformation, using the same primers and protocols asdescribed above in Example 1. FIG. 5A illustrates the relative geneexpression levels for optimized thionin in eight genetically alteredCarrizo lines using RT-qPCR. FIG. 5B illustrates the relative geneexpression levels for chimeric protein in five genetically alteredCarrizo lines using RT-qPCR. FIG. 5C illustrates the relative geneexpression levels for D4E1 protein in four genetically altered Carrizolines using RT-qPCR.

Example 4 Canker Pathogenicity Assays with Genetically Altered CarrizoCitrus

To challenge genetically altered Carrizo, overnight cultures ofXanthomonas citri strain 3213 are centrifuged and diluted to OD₆₀₀ of0.3 with sterile distilled water and further diluted to 10⁴, 10⁵, 10⁶′and 10⁷ CFU/ml, using standard plate-dilution method. (Huang, et al.(1997)) Two leaves of each plant are inoculated with each of the fourbacterial inoculum level. The bacterial suspensions are infiltrated fromthe abaxial side into leaves of the genetically altered Carrizo plantsusing a syringe. Seven genetically altered plants expressing thechimeric protein, nine genetically altered plants expressing optimizedthionin, and four genetically altered plants expressing D4E1 areinoculated, along with two negative control plants. Inoculated plantsare incubated for 10 days, and then the disease development is scoredand photographed. The pathogenicity assay is repeated three times withsimilar results. Results are presented in Table 6, below.

TABLE 6 Bacterial Infiltration Level (CFU/ml) Transgenic Plant Line 10⁴10⁵ 10⁶ 10⁷ Neg. Control 1 ++ +++ +++++ +++++ Neg. Control 2 ++ ++++++++ +++++ Chimera-C3 +/− + +++ +++++ Chimera-C4 − +/− ++ +++++Chimera-C6 − − +/− +++++ Chimera-C9 − − +/− ++++ Chimera-C23 +/− + ++++++++ Chimera-C24 +/− + +++ +++++ Chimera-C29 − + +++ +++++ OptimizedThinion-C1 − +/− + ++++ Optimized Thinion-C3 +/− + +++ +++++ OptimizedThinion-C4 +/− + +++ +++++ Optimized Thinion-C12 − − + ++++ OptimizedThinion-C13 − − +/− ++++ Optimized Thinion-C14 − − +/− ++++ OptimizedThinion-C31 − − ++ ++++ Optimized Thinion-C34 − − + ++++ OptimizedThinion-C41 − − +/− ++++ D4E1-C3 + ++ ++++ +++++ D4E1-C10 ++ +++ ++++++++++ D4E1-C20 + ++ +++ +++++ D4E1-C22 + ++ ++++ +++++ − indicates nocanker observed + indicates canker observed (the number of “+” indicatesseverity of disease observed with “+++++” being highest)

Next, genetically altered Carrizo plants (expressing either optimizedpro-thionin-linker 1-D4E1 or D4E1) are propagated as multiple trees ofeach type alongside non-genetically altered (negative control) plants.All plants are exposed to high ambient levels of Xanthomonas citri in agreenhouse containing infected citrus trees. After three weeks, diseasedevelopment is scored and photographed. The non-genetically alteredplants (negative control) all display signs of severe disease. Manygenetically altered Carrizo plants containing only D4E1 display severedisease, however a few appear somewhat healthy. In contrast, almost allof the genetically altered Carrizo plants expressing optimizedpro-thionin-linker 1-D4E1 are healthy. A few plants display mildsymptoms of canker. See FIG. 6.

Example 5 HLB Pathogenicity Assays with Genetically Altered CarrizoCitrus

To assess the chimeric protein's efficacy in killing CLas in geneticallyaltered Carrizo citrus plants, genetically altered budwood andnon-genetically altered budwood are micrografted to small plants ofstandard citrus rootstocks using standard protocols known to one ofordinary skill in the art. The budwood are obtained from the geneticallyaltered Carrizo citrus plants produced in Example 3 above which exhibitthe best protection against X. citri in Example 4 above. Ten graftedcitrus plants containing budwood from genetically altered plants linesexpressing D4E1 or optimized pro-thionin or the chimeric protein(optimized pro-thionin-linker 1-D4E1) are used in each group along withnegative control group (non-genetically altered budwood grafted plants).Each group is replicated. After the graft union heals and the graftedplants have 3 to 5 leaves, the grafted plants are placed in a no-choiceAsian citrus psyllid (ACP) feeding trial on a lab bench at roomtemperature (˜23° C.) under artificial lighting. Each scion/rootstockcombination are exposed to CLas infected ACP for 2 weeks, after whichthe psyllids are removed. At 3, 6, 9, and 12 weeks after ACP exposure,the entire plant (leaves, stems, and roots) are sacrificed, weighed andcompletely processed for qPCR analysis of CLas titer. Groups ofsimilarly micrografted plants are maintained in the greenhouse for up to2 years and then are planted in the field, monitoring symptoms andgrowth with periodic leaf sampling for CLas titer. All samples arefrozen until processed. DNA is extracted from a subsample (100 mg) offrozen tissue (leaf, stem, and root) using Qiagen DNeasy plant mini kit(Gaithersburg, Md.) using manufacturer's recommended protocols. Todetermine CLas titer levels real-time qPCR is performed, in triplicate,on ABI 7500 real time PCR thermal cycler (Applied Biosystems, FosterCity, Calif.) with Bright Green PCR Master Mix (Sigma-Aldrich, St.Louis, Mo.). Each 25 μL amplification reaction contains 12.5 μL ofBright Green PCR Master Mix, 250 nM of each forward primer and reverseprimer, and 2.0 μL (about 100 ng) DNA template.

The primers used for the real-time qPCR are specific to 16s rDNA forCLas detection (CLas Long=LL): USHRL-LL-F5′-CTTACCAGCCCTTGACATGTATAGG-3′ (forward primer, SEQ ID NO: 63) andUSHRL-LL-R 5′-TCCCTATAAAGTACCCAACATCTAGGTAAA-3′ (reverse primer, SEQ IDNO: 64) or to citrus dehydrin (CD-F 5′-TGAGTACGAGCCGAGTGTTG-3′ (forwardprimer, SEQ ID NO: 65) and CD-R 5′-AAAACTTCACCGATCCACCAG-3′ (reverseprimer, SEQ ID NO: 66)). Citrus dehydrin is used as an internalreference to quantify the number of plant genomes per reaction. Thecycle parameters are 40 cycles of 30 seconds for denaturation at 95° C.and 30 seconds for extension at 60° C., with a melt curve analysis (toverify product identity) as described above in Example 1. Genomeequivalents for CLas and citrus dehydrin are calculated based onstandard curves developed with each primer pair and serial dilutions ofthe target DNA on plasmids of known copy number in a background of cleancitrus DNA. Calculations are based on 3 copies of rDNA target per genomeof CLas and 2 copies of citrus dehydrin per genome. qPCR resultscomparing levels of CLas, and non-parametric symptom data are analyzedusing the non-parametric Kruskal-Wallis test (SAS, Cary, N.C.). Growthdata are analyzed using ANOVA. Standard growth measurements, CLas titerlevels, and disease ratings provide an indication of the potential forcommercially viable growth in the presence of endemic HLB. After thetrees reach fruit bearing age, crop yields will be evaluated todetermine the economic potential of HLB-resistance in the geneticallyaltered grafted citrus plants as compared to standard cultivars.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Alldocuments cited herein are incorporated by reference.

We, the inventors, claim as follows:
 1. A genetically altered plant, a part or progeny thereof comprising a promoter operably linked to a polynucleotide encoding a chimeric protein comprising SEQ ID NO: 8 or SEQ ID NO: 18, wherein said genetically altered plant or parts thereof and its progeny produce said chimeric protein, and wherein said genetically altered plant, part or progeny thereof is more resistant to Xanthomonas citri ssp. citri compared to a wild-type plant's resistance to X. citri ssp. citri.
 2. A method for constructing a genetically altered plant or part thereof having increased resistance to Xanthomonas citri ssp. citri compared to a non-genetically altered plant or part thereof, the method comprising: (i) introducing a polynucleotide encoding a chimeric protein into a plant or part thereof to produce a genetically altered plant or part thereof, wherein said chimeric protein comprising a first domain, a second domain, and a third domain, wherein said first domain comprises optimized thionin or optimized pro-thionin, said second domain comprises D4E1 or pro-D4EI, and said third domain comprises a peptide linker; wherein said peptide linker separates said first domain from said second domain such that said first domain and said second domain can each fold into its appropriate three-dimensional shape and retains its activity, and wherein said peptide linker ranges in length between three amino acids and approximately forty-four amino acids; (ii) selecting a genetically altered plant or part thereof that expresses said chimeric protein, wherein said expressed chimeric protein has anti-bacterial activity against X. citri ssp. citri; wherein said genetically altered plant or part thereof has increased resistance to X. citri ssp. citri compared to the resistance to X. citri ssp. citri of said non-genetically altered plant or part thereof; and wherein said optimized thionin comprises the amino acid sequence of SEP ID NO: 14, said optimized pro-thionin comprises the amino acid sequence of SEP ID NO: 6, said D4E1 comprises the amino acid sequence of SEP ID NO: 2, and said pro-D4EI comprises the amino acid of SEP ID NO:
 62. 3. The method of claim 2, wherein said introducing said polynucleotide encoding said chimeric protein occurs via introgression or transforming said plant with an expression vector comprising said polynucleotide operably linked to a promoter.
 4. The method of claim 2, wherein said optimized thionin or said optimized pro-thionin is located at the amino terminus of said chimeric protein; and wherein said D4E1 is located at the carboxyl terminus of said chimeric protein.
 5. The method of claim 2, wherein said D4E1 or said pro-D4E1 is located at the amino terminus of said chimeric protein; and wherein said optimized thionin is located at the carboxyl terminus of said chimeric protein.
 6. A method of enhancing a wild-type plant's resistance to canker comprising transforming a cell from said wild-type plant with a polynucleotide encoding a chimeric protein to generate a transformed plant cell; and growing said transformed plant cell to generate a genetically altered plant; wherein said chimeric protein comprises a first domain, a second domain, and a third domain; wherein said first domain comprises optimized thionin or optimized pro-thionin, said second domain comprises D4E1 or pro-D4E1, and said third domain comprises a peptide linker; wherein said peptide linker separates said first domain from said second domain such that said first domain and said second domain can each fold into its appropriate three-dimensional shape and retains its activity; wherein said peptide linker is ranges in length between three amino acids and approximately forty-four amino acids; wherein said genetically altered plant or part thereof produces said chimeric protein; and wherein said chimeric protein kills Xanthomonas citri ssp. citri; and wherein said genetically altered plant's resistance to said canker is greater than said wild-type plant's resistance to said canker; and wherein said optimized thionin comprises the amino acid sequence of SEQ ID NO: 14, said optimized pro-thionin comprises the amino acid sequence of SEQ ID NO: 6, said D4E1 comprises the amino acid sequence of SEQ ID NO: 2, and said pro-D4E1 comprises the amino acid of SEQ ID NO:
 62. 7. The method of claim 6, wherein said optimized thionin or said optimized pro-thionin is located at the amino terminus of said chimeric protein; and wherein said D4E1 is located at the carboxyl terminus of said chimeric protein.
 8. The method of claim 6, wherein said D4E1 or said pro-D4E1 is located at the amino terminus of said chimeric protein; and wherein said optimized thionin is located at the carboxyl terminus of said chimeric protein.
 9. A genetically altered plant or part thereof produced by the method of claim
 2. 10. A genetically altered plant or part thereof produced by the method of claim
 6. 11. The method of claim 2, wherein said linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 39, 40, 41, and
 42. 12. The method of claim 11, wherein said chimeric protein comprises an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO:
 18. 13. A genetically altered plant or part thereof produced by the method of claim
 12. 14. The method of claim 6, wherein said linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 39, 40, 41, and
 42. 15. The method of claim 14, wherein said chimeric protein comprises an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO:
 18. 16. A genetically altered plant or part thereof produced by the method of claim
 15. 